Structure of the small G protein Rap2 in a non-catalytic complex with GTP

Abstract

We report a novel crystal form of the small G protein Rap2A in complex with GTP which has no GTPase activity in the crystal. The asymmetric unit contains two complexes which show that a conserved switch I residue, Tyr 32, contributes an extra hydrogen bond to the gamma-phosphate of GTP as compared to related structures with GTP analogs. Since GTP is not hydrolyzed in the crystal, this interaction is unlikely to contribute to the intrinsic GTPase activity. The comparison of other G protein structures to the Rap2-GTP complex suggests that an equivalent interaction is likely to exist in their GTP form, whether unbound or bound to an effector. This interaction has to be released to allow the GAP-activated GTPase, and presumably the intrinsic GTPase activity as well. We also discuss the definition of the flexible regions and their hinges in the light of this structure and the expanding database of G protein structures. We propose that the switch I and switch II undergo either partial or complete disorder-to-order transitions according to their cellular status, thus defining a complex energy landscape comprising more than two conformational states. We observe in addition that the region connecting the switch I and switch II is flexible in Rap2 and other G proteins. This region may be important for protein-protein interactions and possibly behave as a conformational lever arm, as characterized for Arf. Taken together, these observations suggest that the structural mechanisms of small G proteins are significantly driven by entropy-based free energy changes.

RGK Small GTP-binding Proteins Interact with the Nucleotide Kinase Domain of Ca2+-channel β-Subunits via an Uncommon Effector Binding Domain

“…2A). The solved Kir/ Gem-GDP structure is overall very similar to that of the GDPor GTP-bound Rap2A (28), which has 21% sequence identity with Kir/Gem and is the closest homologue to have been crystallized in its GTP-bound form (27). The Switch 1 region in the GDP Kir/Gem structure was not completely resolved, indicating that this region may be disordered, an observation that is confirmed by the recently solved structure of GDP-bound Rad (29).…”

“…The structure of Kir/Gem was taken from the crystallized structure of Gem bound to GDP (PDB 2G2Y). The Switch 1 region ( 95 GVHDSMDDSD-CEVLG 109 ) was rebuilt based on the structure of the GTPbound form of Rap2A as a template (PDB 2RAP) (27). Because Arg-196 is not resolved in PDP 2G2Y, its structure was also rendered.…”

Alternative Splicing of Rac1 Generates Rac1b, a Self-activating GTPase

“…4). In most structures of GppNHp-bound GTPases, the switch II region is well ordered (37), indicating that the insertion in Rac1b contributes to a higher mobility of the switch II region and thus leads to an impaired GTPase reaction of Rac1b (Fig. 1D).…”

Structural mimicry of DH domains by Arfaptin suggests a model for the recognition of Rac–GDP by its guanine nucleotide exchange factors

“…Small GTP‐binding proteins (referred to as G proteins hereafter) form a superfamily of related proteins that alternate between an inactive, GDP‐bound form and an active, GTP‐bound form. A hallmark of G proteins is their conformational plasticity in response to the nature of the bound nucleotide and protein partner (reviewed in [1]). In particular, GDP‐ and GTP‐bound G proteins have different conformations at two regions, called the switch 1 and 2, which specify their recognition by distinct protein partners.…”